Transcription factor NF-KappaB plays a central role in inducing cellular responses to pro-inflammatory signals and controls the expression of a vast array of genes involved in immune response, stress response, cancer and programmed cell death. NF-KappaB is regulated on two levels: Beside cytoplasmic retention by inhibitory molecules (IkappaBs), there is a second regulatory network that controls transcriptional activity of nuclear NF-KappaB complexes. This regulation involves cellular protein kinases and acts through post-translational modifications of NF-KappaB subunits. Specifically, phosphorylation of the NF-KappaB p65 subunit has been implicated as an important step in achieving target gene expression by facilitating promoter transition from its inactive to its active state. The goal of this research program is to elucidate how site specific phosphorylation of p65 regulates nuclear NF-KappaB activity. We provide evidence, that p65 is multiply phosphorylated within the Rel Homology Domain (RHD) and that distinctive phosphorylation regulates its transcriptional activity. Furthermore, we show that p65 phosphorylation controls gene expression in a c/s-element dependent manner. The central hypothesis guiding this proposal is that specificity in NF-KappaB mediated gene expression is achieved through differential phosphorylation of the p65 subunit. Specific Aim #1 will address whether p65 is subject to a "phosphorylation code" that targets p65 transactivation to selected subsets of genes. We will employ molecular and pharmacologic approaches to investigate how promoters containing distinct c/s-acting regulatory elements are responsive to p65 phosphorylation. Specific Aim #2 is to study the relationship between p65 phosphorylation and acetylation. Specific Aim #3 is to explore the mechanism by which p65 RHD phosphorylation controls NF-KappaB transcriptional activity. Studies will employ molecular and biochemical approaches to investigate how differentially phosphorylated p65 proteins affect promoter complex assembly and chromatin remodeling on three model genes. In Specific Aim #4, the hypothesis is tested that p65 differential phosphorylation modulates global gene expression. We will use high-density gene arrays to analyze the impact of differentially phosphorylated p65 proteins on cellular NF-KappaB dependent gene expression. In summary, these studies will establish the role of p65 phosphorylation in achieving signal specificity and provide new insights into the mechanism of NF-KappaB dependent pro-inflammatory gene regulation.